The present invention relates to the field of cancer diagnosis and treatment and, in particular, to determining an expression status of human epidermal growth factor receptor 2 in a biological sample using mass spectrometry.
Adenocarcinoma of the breast is a leading cause of cancer morbidity and mortality among women worldwide. A major challenge faced by clinicians treating patients with breast cancer is how to best assess patient outcome and predict the clinical course of the disease so that the most appropriate treatment regimen can be identified. Determining the expression status of the human epidermal growth factor receptor 2 (HER2) in newly diagnosed breast cancer tissues is critically important for therapeutic decision making. Current guidelines recommend that the HER2 expression status should be evaluated for every patient with newly diagnosed, primary breast cancer.
Unlike most histopathologic testing, which serves as an adjunct to establishing a diagnosis, the results of HER2 testing stand alone in determining which patients afflicted with primary breast cancer are likely to respond to trastuzumab (Herceptin®), a monoclonal antibody directed to HER2. The HER2 expression status can be determined at the DNA-, the mRNA-, or the protein level. Various techniques are available for assessing these different target molecules, each with benefits and drawbacks. Although almost a decade has passed since trastuzumab was initially approved by the USA Food and Drug Administration (FDA), accurate HER2 expression status determination continues to challenge the field of clinical laboratory testing. Currently, two testing methods are approved by the FDA for HER2 testing of breast cancer tissues in the laboratory: immunohistochemical analysis (IHC) and fluorescence in situ hybridization (FISH). Studies using gene transcription profiles have shown that a gene related to human cystein-rich intestinal protein 1 (also referred to as CRIP1) in human breast cancers is similar to non-diseased tissues (Liu et al.: “Thiamine Transporter Gene Expression and Exogenous Thiamine Modulate the Expression of Genes Involved in Drug and Prostaglandin Metabolism in Breast Cancer Cells”. Mol Cancer Res. 2004; 2:477-487; Ma et al.: “Gene Expression Profiles of Human Breast Cancer Progression”. Proc Natl Acad Sci USA. 2003; 100:5974-5979) and other types of cancers. In experiments comparing transcription of the CRIP1 gene to matched normal breast tissue, the mRNA for the target was over-expressed 8 to 10-fold. Two recent studies compared differential gene transcription patterns in HER2-positive and HER2-negative breast cancer cell lines and tissues (Wilson et al.: “Differential Gene Expression Patterns in HER2/Neu-Positive and -Negative Breast Cancer Cell Lines and Tissues”. Am J Pathol. 2002; 161(4):1171-85; Mackay et al.: “cDNA Microarray Analysis of Genes Associated with ERBB2 (HER2/neu) Overexpression in Human Mammary Luminal Epithelial Cells”. Oncogene. 2003; 22(17):2680-8) wherein the mRNA of the CRIP1 gene and HER2-positive were both found to be up-regulated. In contrast to these genetic findings, an increased expression of the CRIP1 at the protein level has not yet been shown.
CRIP1 belongs to the LIM/double zinc finger protein family, which includes cysteine- and glycine-rich protein-1, rhombotin-1, rhombotin-2, and rhombotin-3. Human CRIP1, primarily a cytosolic protein, was cloned in 1997 using RT-PCR from human small intestine RNA and oligonucleotides whose sequence was derived from a human heart homologue of this protein, CRHP.
Mass spectrometry has been widely used to investigate protein patterns in biological samples, such as body fluids or homogenized tissues, to find biomarkers. Interrogation of the resulting complex mass spectrometry (MS) data sets using modern computational tools has provided identifications of both disease-state and patient-prognosis specific protein patterns. In general, biomarkers are used or at least predicted to be used for diagnosing cancer, qualifying different types of cancer, predicting a response to a cancer drug, and predicting a prognosis of a cancer patient. Biomarkers can further be measured and identified with high accuracy using tandem mass spectrometry with, for example, multiple reaction monitoring (MRM).
Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is a powerful tool for investigating protein patterns through direct, in situ, tissue section analyses. Similar to IHC and FISH, MALDI IMS has an advantage over other assay methods (e.g., methods utilizing homogenization) because MALDI IMS is morphologically driven. This allows the direct evaluation of tumor cells, correlation with other morphologic features and the ability to assay smaller patient tumor tissue specimens such as needle core biopsy specimens. MALDI IMS therefore is a seemingly ideal tool for rapid tissue diagnostics and molecular histology. In addition, MALDI IMS can also determine the distribution of hundreds of compounds in a single measurement without labeling.
Some studies have suggested that MALDI IMS provides accurate classifications (Yanagisawa et al.: “Proteomic Patterns of Tumour Subsets in Non-Small-Cell Lung Cancer”, The Lancet, Volume 362, Issue 9382, 9 Aug. 2003, Pages 433-439; Schwartz et al.: “Proteomic-Based Prognosis of Brain Tumor Patients Using Direct-Tissue Matrix-Assisted Laser Desorption Ionization Mass Spectrometry”. Cancer Res. 2005 Sep. 1; 65(17):7674-81). MALDI IMS has been applied to various types of diseased tissues, including human non-small cell lung tumors, gliomas, as well as ovarian and breast cancers. From recent publications it has also become clear that the integration of MALDI IMS into clinical management regarding disease diagnosis and outcome prediction will likely occur in the near future. (Franck et al.: “MALDI IMAGING: State of the Art Technology in Clinical Proteomics”. Mol Cell Proteomics. 2009 May 18; Cornett et al.: “MALDI Imaging Mass Spectrometry: Molecular Snapshots of Biochemical Systems”. Nat Methods. 2007; 4(10):828-33). As MALDI IMS protein expression data becomes available for various tumor tissue types, this approach will provide a common disease-wide methodology that can be applied to a variety of clinical situations. While many of the MALDI IMS studies focus on the identification of new biomarkers, so far only a few studies have evaluated the potential of MALDI IMS for molecular classification of tissues based on protein patterns.